Project Abstract/Summary This is a competitive renewal of NIH grant DK082659 entitled ?Developmental Genes, miRNAs and Adipose Tissue?. Obesity is increasing at epidemic proportions and is a major driving force in the pathogenesis of type 2 diabetes and metabolic syndrome. Over the past decade it has become clear that both white and brown fat are heterogeneous. Thus, accumulation of visceral WAT is associated with insulin resistance and increased risk of metabolic disease, whereas accumulation of subcutaneous WAT may even be protective. This is due, at least in part, to intrinsic, i.e., cell-autonomous, functional differences between adipocytes in these depots. Indeed, recent studies by us and others have shown that there is genetic and functional heterogeneity of white adipocytes within a single depot. Using bioinformatics approaches and single cell cloning, we have now identified lineage tracing markers for at least three classes of white adipocytes in addition to those above. We have also found that different adipose depots differ in their expression of miRNAs and that there are alterations in miRNAs and miRNA processing in adipose tissue in obesity and aging. Furthermore, fat-specific knockout of the miRNA processing enzyme Dicer alters WAT and BAT development, systemic glucose and lipid metabolism, and response to stress. In exciting recent data, we have shown that adipose tissue is a major source of circulating exosomal miRNAs and that these can regulate gene expression in other tissues, forming a novel mode of adipose communication with other tissues. These data have led to two inter-related hypotheses. First, we hypothesize that - in addition to classification of adipose tissue into white, brown and beige fat - white adipocytes themselves are heterogeneous in nature and that this heterogeneity programs differences in mRNA expression and adipocyte function. Secondly, these different classes of white and brown adipocytes differ in their miRNA expression and that a subset of these miRNAs is released in exosomes into the circulation where they act as novel regulators of the effects of fat in other tissues. The specific aims for the next grant period are to: 1) Complete the characterization of white adipocyte heterogeneity using the newly identified lineage markers and expand this using single cell RNAseq to define different subtypes of preadipocyte/adipocytes which differ in function and role in metabolic regulation. 2) Continue to explore the role of adipose tissue derived circulating exosomal miRNAs in metabolism by identifying the molecular signature governing adipocyte miRNA exosomal secretion in vitro and developing an in vivo method for identification of fat- and other tissue-derived circulating exosomal miRNAs. We will also assess exosomal miRNAs in serum of humans with lipodystrophy and obesity and assess the ability of exosomal miRNAs to regulate gene expression in other tissues. Together these data will deepen our understanding of the complex nature of the adipose organ and elucidate the role of circulating adipose-derived exosomal miRNAs as novel adipokines in regulation of metabolism.